ZhaoLan Tang

Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis

Caveolae, 50-100 nm invaginations that represent a subcompartment of the plasma membrane, have been known for many years, but their exact roles remain uncertain. The findings that the caveolae coat protein caveolin is a v-Src substrate and that G-protein-coupled receptors are present in caveolae have suggested a relationship between caveolae, caveolin and transmembrane signalling. The recent isolation of caveolin-rich membrane domains in which caveolin exists as a hetero-oligomeric complex with integral membrane proteins and known cytoplasmic signalling molecules provides support for this hypothesis. Compartmentalization of certain signalling molecules within caveolae could allow efficient and rapid coupling of activated receptors to more than one effector system.

Mutational Analysis of the Properties of Caveolin-1 A NOVEL ROLE FOR THE C-TERMINAL DOMAIN IN MEDIATING HOMO-TYPIC CAVEOLIN-CAVEOLIN INTERACTIONS

Caveolin is a principal structural component of caveolae membranes in vivo Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1. Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes. Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties. Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1ΔC) or the N-terminal domain (Cav-1ΔN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel. Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1. However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes. These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process. To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants. These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer. This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3. Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members. This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, −2, and −3.

Caveolae purification and glycosylphosphatidylinositol-linked protein sorting in polarized epithelia.

Publisher Summary This chapter describes the techniques used for the recombinant expression of glycosylphosphatidylinositol (GPI)-linked proteins in epithelial cell lines and the measurement of cell-surface polarity of endogenous or transfected GPI-linked proteins at steady state and during transport. The chapter also discusses the methods for purification and characterization of caveolae from cultured cells. To study the sorting of endogenous GPI-linked proteins in polarized cells, a series of cell-surface labeling techniques that allow the rapid biochemical determination of the polarity of a given cell-surface antigen is developed. Such labeling techniques depend on the growth of polarized cells on permeable supports that allow for separate access to the apical and basolateral domains. These techniques are then applied to a variety of available intestinal and renal epithelial cell lines, such as the Madin-Darby canine kidney (MDCK), LLC-PK1, Caco-2, and SK-C015 lines, that spontaneously form polarized monolayers in culture. The GPI-linked proteins are detected by their sensitivity to release by treatment with bacterial PI-specific phospholipase C. To measure the polarized sorting of the recombinant proteins during cell-surface transport, additional assays are developed to monitor the cell surface delivery, endocytosis, and transcytosis.

Identification, Sequence, and Expression of an Invertebrate Caveolin Gene Family from the Nematode Caenorhabditis elegans IMPLICATIONS FOR THE MOLECULAR EVOLUTION OF MAMMALIAN CAVEOLIN GENES

Caveolae are vesicular organelles that represent an appendage of the plasma membrane. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Caveolin has been proposed to function as a plasma membrane scaffolding protein to organize and concentrate signaling molecules within caveolae, including heterotrimeric G proteins (α and βγ subunits). In this regard, caveolin interacts directly with Gα subunits and can functionally regulate their activity. To date, three cDNAs encoding four subtypes of caveolin have been described in vertebrates. However, evidence for the existence of caveolin proteins in less complex organisms has been lacking. Here, we report the identification, cDNA sequence and genomic organization of the first invertebrate caveolin gene, Cavce (for caveolin from Caenorhabditis elegans). The Cavce gene, located on chromosome IV, consists of two exons interrupted by a 125-nucleotide intron sequence. The region of Cavce that is strictly homologous to mammalian caveolins is encoded by a single exon in Cavce. This suggests that mammalian caveolins may have evolved from the second exon of Cavce. Cavce is roughly equally related to all three known mammalian caveolins and, thus, could represent a common ancestor. Remarkably, the invertebrate Cavce protein behaves like mammalian caveolins: (i) Cavce forms a high molecular mass oligomer, (ii) assumes a cytoplasmic membrane orientation, and (iii) interacts with G proteins. A 20-residue peptide encoding the predicted G protein binding region of Cavce possesses “GDP dissociation inhibitor-like activity” with the same potency as described earlier for mammalian caveolin-1. Thus, caveolin appears to be structurally and functionally conserved from worms to man. In addition, we find that there are at least two caveolin-related genes expressed in C. elegans, defining an invertebrate caveolin gene family. These results establish the nematode C. elegans as an invertebrate model system to study caveolae and caveolin in vivo.

The primary sequence of murine caveolin reveals a conserved consensus site for phosphorylation by protein kinase C.

We report here the cloning of the murine cDNA encoding caveolin, a known v-Src substrate and caveolar marker protein. Interestingly, analysis of the murine cDNA and comparison with caveolin from other species reveals a previously unrecognized consensus site for protein kinase C (PKC) phosphorylation. This finding could have important implications as (i) both the morphology and function of caveolae are dramatically affected by PKC activators; and (ii) PKC alpha is concentrated in isolated caveolin-rich membrane domains. In addition, this first step should facilitate the use of the mouse as a genetic system for elucidating the role of caveolin in caveolar functioning.

Caveolae and human disease: functional roles in transcytosis, potocytosis, signalling and cell polarity

Caveolae are 50–100 nm invaginations that represent a sub-compartment of the plasma membrane. Recent studies have implicated these membranous structures in: (1) transcytosis of macromolecules (such as LDL and AGEs) across capillary endothelial cells; (2) potocytic uptake of small molecules via GPI-linked receptors coupled with an unknown anion transport protein; (3) certain transmembrane signalling events; and (4) polarized trafficking of GPI-linked proteins in epithelial cells. Biochemical isolation and characterization of these domains reveals the molecular components that could perform these diverse functions: scavenger receptors for oxidized LDL and AGEs, namely CD 36 and RAGE, respectively (transcytosis); plasma membrane porin (potocytosis); heterotrimeric G-proteins and Src-like kinases (signalling); and Rap GTPases (cell polarity). As such, these findings have clear implications for understanding the molecular pathogenesis of several human diseases — including atherosclerosis, diabetic vascular complications, and cancerous cell transformations.

The apical sorting of glycosylphosphatidylinositol-linked proteins

Publisher Summary Many cells, from yeast to human, use glycosylated form of phosphatidylinositol to anchor proteins to the cell surface. Several terms have been coined to describe this anchoring mechanism. They include “glycosylphosphatidylinositol (GPI) anchoring,” “glypiation,” “phosphatidylinositol-glycan (PIG) tailing,” and “greasy foot.” All GPI anchors contain a conserved glycan core structure, composed of ethanolamine, phosphate, mannose, glucosamine, and inositol, whereas GPI-linked proteins are found clustered in caveolae, a specialized domain of the plasma membrane. This chapter discusses the reasons for GPI-linked proteins being selectively transported to the apical surface of polarized epithelial cells, with GPI acting as a dominant apical trafficking signal.

Caveolae: Portals for transmembrane signaling and cellular transport

Publisher Summary Caveolae, also known as “plasmalemmal vesicles,” are 50–100nm flask-shaped vesicular organelles located at the plasma membrane and within the Golgi complex. Caveolae play a central role in normal and pathogenic signaling events and represent a focal point for a variety of human diseases. Caveolin was first identified as a major v-Src substrate and later as a caveolar marker protein. Known to be an integral membrane phosphoprotein, caveolin could play the role of an adaptor molecule, linking other caveolar components to cytoplasmic signaling molecules. The role of caveolae in the transcellular transport of protein was studied in endothelial cells. A series of both large and small fluid-phase tracer molecules were transported across capillary endothelial cells via caveolae.